The invention relates to microbial spores treated with various additives in order to alter sensitivity to sterilants.
Biological indicators (BIs) have been used to test and/or determine the effectiveness of sterilization processes. Typically, biological indicators containing microbial spores are exposed to a selected sterilant or sterilizing process and then the survival of the exposed spores is determined by placing the exposed spores in an environment capable of sustaining germination and outgrowth of spores. Microbial spores are typically more resistant to sterilization processes than most types of microorganisms and it is assumed that a sterilization process that will kill microbial spores also will kill any contaminating microorganisms.
Traditional BIs based on growth could not be used to measure directly a sterilization assurance level as low as about 10.sup.-1 and generally required incubation periods of at least two days and up to seven days before the effectiveness of the sterilization process could be assessed. The development of linear reaction velocity (LRV) technology has facilitated a sensitive method for the rapid determination of sterilization effectiveness over a wide range of frequency of survival from 10.sup.9 to as low as 10.sup.-16 viable spores per unit. As spores germinate, they absorb water and lose the capability of scattering light in spore-containing suspensions. This property allows the germination process to be followed spectrophotometrically as a decrease in light absorption or a decrease in light scattering. To determine spore germination rates, i.e., the decrease in absorbance per unit time, germination kinetics curves can be created by plotting the absorbance at 480 nm ("Abs.sub.480 ") of a germinating spore suspension as a function of time. After spore germination is initiated, there is a lag period where little or no change in absorbance is observed. When a detectable percentage of the spores begin to germinate, a decrease in the Abs.sub.480 of the spore suspension is observed. The decrease in the Abs.sub.480 of the spore suspension is recorded until a majority of the spores germinate. The observed rate is affected by both the number of spores germinating and the time needed for a spore to complete germination. Thus, the more synchronous the spore germination of a given spore population, the higher the germination rate.
The LRV is the maximum spore germination rate for a particular population of spores in a particular germination medium. The LRV is computed from the descending linear portion of the germination kinetics curve that follows the lag period. The LRV is presented as the absolute value of the slope of the descending linear portion of the germination kinetics curve. Generally, LRV is expressed in units Abs.sub.480 /min. Depending on the condition of the spores and the type of germination medium used, the lag period may vary and thus the time interval representing the descending linear portion may vary. After exposing spores to a sterilant, LRVs correlate with the survival of viable spores or cells in a linear relationship. The lower the LRV, the lower the probability of non-sterile units being present in a given biological load that was subjected to a sterilization process. See, WO 95/21936, filed Feb. 15, 1995.
It has been observed that death of microorganisms within a population due to an external factor, such as heat or gas sterilants, is described best using first order kinetics, since the decrease in the number of such organisms is logarithmic. See, for example, Pflug, I. J. and R. G. Holcomb, "Principles of the thermal destruction of microorganisms", In Disinfection, Sterilization, and Preservation, Fourth Edition, S. S. Block, ed., Lea and Febiger, (1991), pp. 83-128. Thus, the number of organisms surviving per unit after increasingly longer exposure to a sterilant or killing treatment may be determined using the following linear regression equation (equation 1) and then plotting the calculated data on semilog graph paper. EQU log N=-U/D+log N.sub.0 (eq. 1)
U is equal to the number of minutes of sterilant exposure. N.sub.0 is equal to the number of spores or cells per unit at the beginning of the sterilization process. N is equal to the number of microorganisms remaining per unit after sterilant exposure for a given time, U. D is a decimal reduction time (specifically, minutes required to kill one log of spores or cells), which is a constant for a given set of conditions and a given batch or crop of spores or cells. Thus, D is the negative reciprocal of the slope of a straight-line death curve.
Read-out reliability (ROR) is defined as the ratio of the number of positive BIs after two days of growth compared to the number of positive BIs after 7 days of growth. For a read-out of sterilization results earlier than 7 days to be valid, an ROR of at least 97% is required. Shortening readout time is highly desirable and would enhance the effectiveness of the BI assay.
Different methods of sterilization require spores with defined levels of resistance, which likewise give rise to different D values. Not all organisms can achieve the required D values for a particular sterilization method. For example, spores from Bacillus subtilis are best suited for ethylene oxide (EtO) sterilization, whereas spores from Bacillus stearothermophilus are best suited for high temperature steam sterilization. For other sterilization methods such as hydrogen peroxide plasma, the most suitable organism has not yet been identified. It would be useful if spore resistance to a certain sterilization method could be altered to allow use of an organism when sterilization conditions change or when it is more economical to produce spores from a particular organism whose native resistance to a particular sterilization process may not be optimal.